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Acridine compounds have been described as promising anticancer agents. Previous studies showed that (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a spiro-acridine compound, has antitumor activity on Ehrlich tumor and low toxicity. Herein, we investigated its antitumor effect against human cells in vitro.MTT assay was used to assess cytotoxicity of AMTAC-06 (3.125-200 µM) against tumor and non-tumor cells, and the half-maximal inhibitory concentration (ICAMTAC-06 showed higher cytotoxicity against colorectal carcinoma HCT-116 cells (ICAMTAC-06 presents anticancer activity against HCT-116 cells by regulating the cell cycle, inducing apoptosis and an antioxidant action.

BACKGROUND/AIM Studies with acridine compounds have reported anticancer effects. Herein, we evaluated the toxicity and antitumor effect of the (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a promising anticancer spiro-acridine compound. MATERIALS AND METHODS The toxicity of AMTAC-06 was evaluated on zebrafish and mice. Antitumor activity was assessed in Ehrlich ascites carcinoma model. Effects on angiogenesis, cytokine levels and cell cycle were also investigated. RESULTS AMTAC-06 did not induce toxicity on zebrafish and mice (LD50 approximately 5000 mg/kg, intraperitoneally). No genotoxicity was observed on micronucleus assay. AMTAC-06 significantly reduced the total viable Ehrlich tumor cells and increased sub-G1 peak, suggesting apoptosis was triggered. Moreover, the compound significantly decreased the density of peritumoral microvessels, indicating an anti-angiogenic action, possibly dependent on the cytokine modulation (TNF-α, IL-1β and IFN-γ). No significant toxicological effects were recorded for AMTAC-06 on tumor transplanted animals. CONCLUSION AMTAC-06 has low toxicity and a significant antitumor activity.

Microbiota, the microorganisms that colonize living organisms. Oysters rely exclusively on an innate immune system. This study characterized the cultivable gastrointestinal tract microbiota (GTM) of the oyster Crassostrea gasar and evaluated their influence on immune responses. Antibiotics were used to reduce or alter the microbiota during in vitro and in vivo assays. Haemocyte viability, concentration, and phagocytic capacity, as well as the production of reactive oxygen species (ROS), were evaluated in antibiotic-treated and control oysters. Microbiological analysis of the gastrointestinal tract was also performed; bacteria were selected using culture media and were identified by 16S ribosomal DNA. The results showed that the antibiotics eliminated bacteria in vitro but increased their concentrations in vivo . The GTM was composed of 26% amylolytic bacteria, 21% lipolytic bacteria, 18% proteolytic bacteria, 18% cellulolytic bacteria and 17% lactic acid bacteria; there were no differences in the amounts of these bacterial types between the control and treated oysters. Three major bacterial phyla, Proteobacteria, Firmicutes and Actinobacteria, and seven genera, Labrenzia , Pseudomonas , Halomonas , Shewanella , Vibrio , Bacillus , and Micrococcus , were detected. The concentration, viability, and phagocytic capacity of hemocytes and the production of ROS were not significantly altered by antibiotic treatment. In conclusion, the antibiotics altered the number of heterotrophic bacteria without changing hemocyte function, suggesting that this assay could be useful for verifying the influence of microbiota in host-parasite interactions.

Oyster production is an economic activity of great interest worldwide. Recently, oysters have been suffering significant mortalities from OsHV-1infection, which has resulted in substantial economic loses in several countries around the world. Understanding viral pathogenicity mechanisms is of central importance for the establishment of disease control measures. Thus, the present work aimed to identify and characterize miRNAs from OsHV-1 as well as to predict their target transcripts in the virus and the host. OsHV-1 genome was used for the in silico discovery of pre-miRNAs. Subsequently, viral and host target transcripts of the OsHV-1 miRNAs were predicted according to the base pairing interaction between mature miRNAs and mRNA 3' untranslated regions (UTRs). Six unique pre-miRNAs were found in different regions of the viral genome, ranging in length from 85 to 172 nucleotides. A complex network of self-regulation of viral gene expression mediated by the miRNAs was identified. These sequences also seem to have a broad ability to regulate the expression of host immune-related genes, especially those associated with pathogen recognition. Our results suggest that OsHV-1 encodes miRNAs with important functions in the infection process, inducing self-regulation of viral transcripts, as well as affecting the regulation of Pacific oyster transcripts related to immunity. Understanding the molecular basis of host-pathogen interactions can help mitigate the recurrent events of oyster mass mortalities by OsHV-1 observed worldwide.

International audience; Disseminated neoplasia (DN) is a disease that affects bivalves worldwide and can lead to mass mortalities. In the present study, a pathological survey conducted from December 2011 to August 2012 in Crassostrea gasar, an oyster of commercial interest in northeast Brazil, revealed the occurrence of DN in oysters reared in the Mamanguape estuary, Paraiba State, Brazil. The present work describes the pathological and functional aspects of the disease in C. gasar by light microscopy (haemolymph cell monolayer and histological section) and flow cytometry analyses. The prevalence of the disease was low (7.1% of 182 oysters examined). Enlarged (neoplastic) cells showed reniform, ovoid or circular-shaped nuclei, with prominent nucleoli and predominantly short filipodia. They were found in the haemolymph and infiltrated the connective tissues of different organs, including the digestive system, gills and gonads, as well as in the sinuses and vessels. Three levels of progression of DN in tissues were observed, light (61.5%), moderate (15.4%) and advanced (23.1%). The viability of neoplastic cells circulating in the haemolymph (97.4%) was similar to that in the haemocytes (95.7%). The neoplastic cells showed low phagocytic ability (3.9%) compared with that of haemocytes (42.4%). Conversely, reactive oxygen species production (679 A.U.) and the total haemocyte count (3.9 x 10(6) cells mL(-1)) were higher in the affected oysters than in unaffected oysters (268 A.U. and 1.5 x 10(6) cells mL(-1), respectively). The low prevalence and primarily mild intensity found in the sampled oysters does not preclude an impact at the population level. A timely survey of DN is thus recommended in order to assess the severity and impact of this disease in wild and cultured populations of C. gasar oysters.

Few studies have associated the effects of changes in caloric intake and redox disturbances in the gastrointestinal tract. Therefore, the present study aimed at evaluating the hypercaloric diet consumption influence on the contractile reactivity of intestinal smooth muscle, morphology, and oxidative stress of rat ileum. Wistar rats were randomly divided into groups that received a standard diet and fed with a hypercaloric diet for 8 weeks. Animals were euthanized, and the ileum was isolated to isotonic contraction monitoring. Morphology was evaluated by histological staining and oxidative stress by quantification of malondialdehyde levels and total antioxidant activity. Cumulative concentration-response curves to KCl and carbachol were attenuated in rats fed with a hypercaloric diet compared to those that received a standard diet. In addition, an increase in caloric intake promotes a rise in the thickness of the longitudinal smooth muscle layer of rat ileum and tissue malondialdehyde levels, characterizing lipid peroxidation, as well as a decrease in the antioxidant activity. Thus, it was concluded that the consumption of a hypercaloric diet impairs rat intestinal contractility due to mechanisms involving modifications in the intestinal smooth muscle architecture triggered by redox disturbances.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 μM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 μM), for 24–48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 μM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 μM after 24 h and at 10 μM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

2,4-Dichlorophenoxyacetic acid (2,4-D) herbicide is the main ingredient in over 1500 commercially available products such as Weedestroy® AM40 and DMA® 4 IVM. Although the liver has been identified as one of the organs that are affected by this herbicide, reports on its hepatotoxic effects available in the literature are restricted to rats. Thus, there is a gap in information on other organisms that may be vulnerable to 2,4-D exposure, such as fish. Therefore, the present work aimed to assess the hepatotoxic potential of 2,4-D in fish using zebrafish (Danio rerio) larvae as a model system. For this purpose, its acute toxicity to zebrafish embryos was assessed, as well as its sublethal effects (

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Human activities have caused climate changes and altered the salinity of the oceans. The reduction of the salinity is one of the factors that may limit the distribution and the survival of marine organisms. Coelomocytes are the immune system cells of the echinoderms and have been studied as biomarkers of stress. The aim of the present study was to investigate the effect of the salinity on the immune system cells of the tropical sea urchin Echinometra lucunter . Animals were collected in Joao Pessoa coast (Brazilian Northeast). Animals or coelomocytes were exposed to different salinities (25, 35, 45) from 4 to 24 h. Phagocytic parameters, production of reactive oxygen species (ROS), mitochondrial activity and ABC transporter activity were analyzed. The phagocytic parameters did not change when animals or cells were exposed to a salinity of 25 or 45 in any time intervals monitored. However, the coelomocytes concentration increased when animals were exposed to the lower salinity. The levels of ROS were higher when cells were incubated at a salinity of 25 but lower when cells were kept at a salinity of 45. It was observed the loss of the mitochondrial inner membrane potential when coelomocytes were incubated at a salinity of 45. The activity of ABC transporters decreased when cells were incubated at the lowest salinity and increased when cells were incubated at the highest salinity tested. The present work shows that the immune system of the tropical sea urchins Ei lucunter tolerates salinity changes from 25 to 45, and suggests two cellular parameters (ROS levels and ABC transporters activity) as potential biomarkers for the monitoring of the impact of environmental salinity changes.

Asthma is a chronic inflammatory disease of the airways characterized by immune cell infiltrates, bronchial hyperresponsiveness, and declining lung function. Thus, the possible effects of virgin coconut oil on a chronic allergic lung inflammation model were evaluated. Morphology of lung and airway tissue exhibited peribronchial inflammatory infiltrate, epithelial hyperplasia, and smooth muscle thickening in guinea pigs submitted to ovalbumin sensitization, which were prevented by virgin coconut oil supplementation. Additionally, in animals with lung inflammation, trachea contracted in response to ovalbumin administration, showed a greater contractile response to carbachol (CCh) and histamine, and these responses were prevented by the virgin coconut oil supplementation. Apocynin, a NADPH oxidase inhibitor, did not reduce the potency of CCh, whereas tempol, a superoxide dismutase mimetic, reduced potency only in nonsensitized animals. Catalase reduced the CCh potency in nonsensitized animals and animals sensitized and treated with coconut oil, indicating the participation of superoxide anion and hydrogen peroxide in the hypercontractility, which was prevented by virgin coconut oil. In the presence of L-NAME, a nitric oxide synthase (NOS) inhibitor, the CCh curve remained unchanged in nonsensitized animals but had increased efficacy and potency in sensitized animals, indicating an inhibition of endothelial NOS but ineffective in inhibiting inducible NOS. In animals sensitized and treated with coconut oil, the CCh curve was not altered, indicating a reduction in the release of NO by inducible NOS. These data were confirmed by peribronchiolar expression analysis of iNOS. The antioxidant capacity was reduced in the lungs of animals with chronic allergic lung inflammation, which was reversed by the coconut oil, and confirmed by analysis of peribronchiolar 8-iso-PGF2α content. Therefore, the virgin coconut oil supplementation reverses peribronchial inflammatory infiltrate, epithelial hyperplasia, smooth muscle thickening, and hypercontractility through oxidative stress and its interactions with the NO pathway.

International audience; Bivalves were long thought to be “symptomless carriers” of marine microalgal toxins to human seafood consumers. In the past three decades, science has come to recognize that harmful algae and their toxins can be harmful to grazers, including bivalves. Indeed, studies have shown conclusively that some microalgal toxins function as active grazing deterrents. When responding to marine Harmful Algal Bloom (HAB) events, bivalves can reject toxic cells to minimize toxin and bioactive extracellular compound (BEC) exposure, or ingest and digest cells, incorporating nutritional components and toxins. Several studies have reported modulation of bivalve hemocyte variables in response to HAB exposure. Hemocytes are specialized cells involved in many functions in bivalves, particularly in immunological defense mechanisms. Hemocytes protect tissues by engulfing or encapsulating living pathogens and repair tissue damage caused by injury, poisoning, and infections through inflammatory processes. The effects of HAB exposure observed on bivalve cellular immune variables have raised the question of possible effects on susceptibility to infectious disease. As science has described a previously unrecognized diversity in microalgal bioactive substances, and also found a growing list of infectious diseases in bivalves, episodic reports of interactions between harmful algae and disease in bivalves have been published. Only recently, studies directed to understand the physiological and metabolic bases of these interactions have been undertaken. This review compiles evidence from studies of harmful algal effects upon bivalve shellfish that establishes a framework for recent efforts to understand how harmful algae can alter infectious disease, and particularly the fundamental role of cellular immunity, in modulating these interactions. Experimental studies reviewed here indicate that HABs can modulate bivalve-pathogen interactions in various ways, either by increasing bivalve susceptibility to disease or conversely by lessening infection proliferation or transmission. Alteration of immune defense and global physiological distress caused by HAB exposure have been the most frequent reasons identified for these effects on disease. Only few studies, however, have addressed these effects so far and a general pattern cannot be established. Other mechanisms are likely involved but are under-studied thus far and will need more attention in the future. In particular, the inhibition of bivalve filtration by HABs and direct interaction between HABs and infectious agents in the seawater likely interfere with pathogen transmission. The study of these interactions in the field and at the population level also are needed to establish the ecological and economical significance of the effects of HABs upon bivalve diseases. A more thorough understanding of these interactions will assist in development of more effective management of bivalve shellfisheries and aquaculture in oceans subjected to increasing HAB and disease pressures.

SUMMARYField and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

The invasive bivalve Mytilopsis sallei caused several economic and ecological impacts in Asia. In Brazil, M. sallei was first reported in 2004, and thus far, little is known about its ecological impact on Brazilian estuaries. The present study aims to evaluate the abundance and distribution of M. sallei and to analyse the environmental factors that led to the establishment of this species in a Brazilian Northeast estuary. Sampling was carried out in six stations along the estuary, covering a broad spectrum of salinities. Results showed that M. sallei was the most abundant benthic species in this estuary, although it was not broadly distributed, occurring only in one station (S1), the most polluted and the one with the lowest salinities (mean 17.3 ± 11.56), which was the abiotic factor controlling the distribution of M. sallei. This station also had the lowest diversity of species (H′ = 0.3269). The main M. sallei microhabitat was the pneumatophores roots from mangrove trees and decomposing wood. Despite the high abundance of M. sallei in S1, this species experienced negative effect of other benthic species suggesting that its distribution was limited by the high abundance of native species in other stations. Our results suggest that the distribution of M. sallei is influenced by a set of characteristics: preference for low salinity, favourable substrate type, tolerance to pollutants, and less direct competitors. Understanding how invasive species are distributed and relate to native species will provide more robust strategies to help maintain environmental health that ultimately will prevent the expansion of M. sallei population.

The production of native oysters Crassostrea rhizophorae and Crassostrea gasar is expanding in northeastern Brazil, where reports of bivalve pathology are scarce. Recently, Perkinsus marinus was detected for the first time infecting C. rhizophorae in the state of Paraiba, in northeastern Brazil. Thus, more information on factors affecting the health of oysters is needed, especially concerning the pathogens listed by the World Organisation for Animal Health (OIE). The current study evaluated the health status of wild and cultured C. gasar mangrove oysters from the estuary of the Rio Sao Francisco, Sergipe state, northeastern Brazil. Results show that C. gasar oysters cultivated in the Rio Sao Francisco estuary had a greater prevalence of diseases and pathogens than oysters from a natural population in the same estuary, including maladie du pied (shell disease), Rickettsia—like organisms in gills, Steinhausia sp. oocyte infections, and Polydora sp. valve infestations. Of the two OIE notifiable diseases investigated, perkinsosis occurred at variable levels of prevalence that were always greater among cultured oysters (50.8%) than wild oysters (25.8%), and the intensity was predominantly low, suggesting low mortality impact. Bonamiosis was not detected.

Crassostrea rhizophorae and C. gasar oysters are cultivated in the northeast region. Perkinsus parasites infect bivalves, and their effects on oysters from tropical regions are poorly understood. This study evaluated the impact of Perkinsus infection on the productive traits of native oysters. Oysters were sampled bimonthly during 7 months, from July 2010 to February 2011, to evaluate growth rate, mortality and shell color patterns (white and dark-gray) (n = 500), and to determine the prevalence and intensity of Perkinsus (n = 152). Perkinsus and Crassostrea species were determined using molecular tools. Results showed that most dark-gray (90%, n = 20) and white (67%, n = 18) oysters were C. gasar and C. rhizophorae, respectively. Oysters showed a high growth rate and moderate cumulative mortality (44%). C. gasar oysters grew better and showed lower mortality and lower incidence of Perkinsus compared to C. rhizophorae. The mean prevalence of Perkinsus was moderate (48%), but the infection intensity was light (2.2). Perkinsosis affected very small oysters (19.4 mm). In conclusion, native oysters, especially C. gasar, have a great potential for culture, mortality is not associated with perkinsosis, and the shell color of oysters can be used to improve selection for spats with better performance. Resumo Crassostrea rhizophorae e C. gasar são cultivadas na região Nordeste. Parasitas Perkinsus infectam bivalves e seus efeitos em ostras de regiões tropicais são pouco compreendidos. Este estudo avaliou o impacto da infecção por Perkinsus em parâmetros de produção de ostras nativas. Ostras foram coletadas bimestralmente durante 7 meses, de julho de 2010 a fevereiro de 2011, para avaliar crescimento, mortalidade e padrão de coloração da concha (branca e cinza-escura) (n = 500); além da presença e intensidade de Perkinsus (n = 152). Perkinsus e Crassostrea foram identificados por abordagem molecular. Os resultados mostraram que as ostras cinza-escuras (90%, n = 20) e brancas (67%, n = 18) eram C. gasar e C. rhizophorae, respectivamente. As ostras mostraram uma boa taxa de crescimento e mortalidade acumulada moderada (44%). C. gasar cresceu melhor com menor mortalidade e menor incidência de Perkinsus que C. rhizophorae. A prevalência média de Perkinsus foi moderada (48%), mas a intensidade de infecção foi leve (2,2). A perkinsiose afetou ostras pequenas (19,4 mm). Em conclusão, ostras nativas, especialmente C. gasar, têm grande potencial de produção; sem mortalidade associada à perkinsiose; e, a cor da concha pode ser usada para melhorar a seleção de sementes com melhor desempenho.

Hemocyte populations of the pearl oyster Pteria hirundo were characterized at morphological, ultrastructural and functional levels. Three main hemocyte populations were identified: hyalinocytes, granulocytes and blast-like cells. Hyalinocytes were the most abundant population (88.2%) characterized by the presence of few or no granules in the cytoplasm and composed by two subpopulations, large and small hyalinocytes. Comparatively, granulocytes represented 2.2% of the hemocyte population and were characterized by the presence of numerous large electron-lucid granules in the cytoplasm. Finally, the blast-like cells (9.5%) were the smallest hemocytes, showing spherical shape and a high nucleus/cytoplasm ratio. Hemocytes exhibited a significant phagocytic capacity for inert particles (38.5%) and showed to be able to produce microbicidal molecules, such as reactive oxygen species (ROS) (ex vivo assays). The immune role of hemocytes was further investigated in the P. hirundo defense against the Gram-negative Vibrio alginolyticus. A significant decrease in the total number of hemocytes was observed at 24 h following injection of V. alginolyticus or sterile seawater (injury control) when compared to naive (unchallenged) animals, indicating the migration of circulating hemocytes to the sites of infection and tissue damage. Bacterial agglutination was only observed against Gram-negative bacteria (Vibrio) but not against to marine Gram-positive-bacteria. Besides, an increase in the agglutination titer was observed against V. alginolyticus only in animals previously infected with this same bacterial strain. These results suggest that agglutinins or lectin-like molecules may have been produced in response to this particular microorganism promoting a specific recognition. The ultrastructural and functional characterization of P. hirundo hemocytes constitutes a new important piece of the molluscan immunity puzzle that can also contribute for the improvement of bivalve production sustainability.

Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus. In Brazil, two species, P. beihaiensis and P. marinus, are frequently found infecting native oysters ( Crassostrea gasar and C. rhizophorae ) from cultured and wild populations in several states of the Northeast region. The impacts of this disease in bivalves from Brazil, as well as the interactions with environmental factors, are poorly studied. In the present work, we evaluated the in vitro effects of the cyanobacteria Synechocystis spp. on trophozoites of P. marinus and haemocytes of C. gasar . Four cyanobacteria strains isolated from the Northeast Brazilian coast were used as whole cultures (WCs) and extracellular products (ECPs). Trophozoites of P. marinus were exposed for short (4 h) and long (48 h and 7 days, the latter only for ECPs) periods, while haemocytes were exposed for a short period (4 h). Cellular and immune parameters, i.e. cell viability, cell count, reactive oxygen species production (ROS) and phagocytosis of inert (latex beads) and biological particles (zymosan and trophozoites of P. marinus ) were measured by flow cytometry. The viability of P. marinus trophozoites was improved in response to WCs of Synechocystis spp., which could be a beneficial effect of the cyanobacteria providing nutrients and reducing reactive oxygen species. Long-term exposure of trophozoites to ECPs of cyanobacteria did not modify in vitro cell proliferation nor viability. In contrast, C. gasar haemocytes showed a reduction in cell viability when exposed to WCs, but not to ECPs. However, ROS production was not altered. Haemocyte ability to engulf latex particles was reduced when exposed mainly to ECPs of cyanobacteria; while neither the WCs nor the ECPs modified phagocytosis of the biological particles, zymosan and P. marinus . Our results suggest a negative effect of cyanobacteria from the Synechocystis genus on host immune cells, in contrast to a more beneficial effect on the parasite cell, which could together disrupt the balance of the host-parasite interaction and make oysters more susceptible to P. marinus as well as opportunistic infections.

Bivalves are filter feeders that obtain food from seawater that may contain infectious agents, such as the protozoan parasites Perkinsus marinus and P. olseni that are associated with massive mortalities responsible for losses in the aquaculture industry. Despite all physical and chemical barriers, microorganisms cross epithelia and infect host tissues to cause pathologies. Epigenetics mechanisms play important roles in a variety of human processes, from embryonic development to cell differentiation and growth. It is currently emerging as crucial mechanism involved in modulation of host-parasite interactions and pathogenesis, promoting discovery of targets for drug treatment. In bivalves, little is known about epigenetic mechanism in host parasite interactions. The objective of the present study was to evaluate the effect of Perkinsus sp. infections on DNA methylation levels in tissues of Crassostrea gasar oysters. Samples were collected in 2015 and 2016 in the Mamanguape River estuary (PB). Oyster gills were removed and used for Perkinsus sp. diagnosis. Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n = 60) and infected oysters (n = 60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

WOS:000416186800021; International audience; ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraiba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes \textgreater colorless spherulocytes \textgreater vibrate cells \textgreater red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C gigas. Additionally, our results showed that C gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Blooms of the harmful alga Dinophysis acuminata, which produces okadaic acid (OA), are becoming recurrent in Santa Catarina coast, where most of the shellfish marine farms in Brazil are located. We evaluated the impact of D. acuminata blooms on various haemato-immunological parameters and on tissue integrity of cultivated oysters (Crassostrea gigas) and mussels (Perna perna). Animals were sampled during two natural algal blooms, one at Praia Alegre (PA: 2950 cells l−1) and the other at Praia de Zimbros (PZ: 4150 cells l−1). Control animals were sampled at the same sites, 30 days after the end of the bloom. The assayed parameters were: total (THC) and differential (DHC) haemocyte counts, percentage of apoptotic haemocytes (AH), phenoloxidase activity (PO), agglutinating titre (AT) and total protein concentration in haemolymph (PC). Histological analyses were carried out in oysters from PZ. The results showed that some immune parameters were modulated during the toxic blooms, but not in a consistent manner, especially in mussels that accumulated more OA (10×) than oysters. For example, mussel THC decreased significantly (54%) during the bloom at PA, whereas it augmented markedly (64%) at PZ. PO activity was significantly altered by the algal blooms in both bivalve species, while PC increased significantly (66%) only in mussels from PZ bloom. The other parameters (DHC, AH and AT) did not vary in both bivalve species. Histological analyses showed an intense haemocytic infiltration throughout the oyster digestive epithelium, particularly into the stomach lumen during the algal bloom.

Tumor cells have specific features, including angiogenesis induction, cell cycle dysregulation, and immune destruction evasion. By inducing a T helper type 2 (Th2) immune response, tumor cells may favor immune tolerance within the tumor, which allows progression of cancer growth. Drugs with potential antitumor activity are the spiro-acridines, which is a promising new class of acridine compounds. Herein, the novel spiro-acridine (E)-5′-oxo-1′-((3,4,5-trimethoxybenzylidene)amino)-1′,5′-dihydro-10H-spiro[acridine-9,2′-pyrrole]-4′-carbonitrile (AMTAC-17) was synthesized and tested for antitumor effects. Toxicity evaluation was performed in mice after acute treatment (2000 mg/kg, intraperitoneally, i.p.). The Ehrlich ascites carcinoma model was used to investigate the antitumor activity of AMTAC-17 (12.5, 25, or 50 mg/kg, i.p.) after seven days of treatment. Effects on the cell cycle, angiogenesis, and inflammatory responses were investigated. LD50 (lethal dose 50%) was estimated to be higher than 5000 mg/kg. AMTAC-17 reduced the Ehrlich tumor’s total viable cancer cells count and peritumoral micro-vessels density, and induced an increase in the sub-G1 peak. Additionally, there was an increase of Th1 cytokine profile levels (IL-1β, TNF-α, and IL-12). In conclusion, the spiro-acridine compound AMTAC-17 presents low toxicity, and its in vivo antitumor effect involves modulation of the immune system to a cytotoxic Th1 profile and a reduction of tumor angiogenesis.

International audience; Blood cells or hemocytes are the major component of the cellular immune responses in bivalves. Recent evidences have demonstrated that the immune functions of these cells can be impaired by harmful algae and their toxins. In this study, we compared the in vitro effect of the toxic dinoflagellate Alexandrium minutum and its purified paralytic toxin, saxitoxin (STX) on the oyster Crassostrea gigas hemocytes. Oyster hemocytes were incubated with A. minutum or STX, and their size, complexity, viability, phagocytic activity and reactive oxygen species (ROS) production were analyzed through flow cytometry. Also, expression of eleven defense-related genes was evaluated through real-time quantitative PCR. Cell incubation with both, the noxious microalga and STX did not alter hemocyte viability neither complexity. However, their phagocytic capacity and ROS production were significantly affected by both treatments. On the other hand, only A. minutum exposure resulted in increase of hemocyte size. Transcript levels of the gene encoding the cytokine Interleukin-17 was significantly higher (4.5-fold) after hemocytes were exposed to A. minutum, which contrasted with a decreased (7-fold) number of transcripts, when hemocytes were treated with STX. The purified toxin also led to lower transcript levels of a cytochrome P450 gene, the CYP356A1 (3.5-fold). On the other hand, hemocytes incubated with STX presented higher mRNA levels of antimicrobial peptide defensin-2 (7-fold) and heat shock protein Hsp70 (2-fold) genes. Altogether, these results suggest that the noxious alga A. minutum and STX can indeed negatively affect immunocompetence of C. gigas hemocytes, although in distinct manners. These differences could be due to a lower STX bioavailability in living microalgal cultures or alternatively to the presence of allelopathic compounds produced by living microalgae. This is the first report comparing the transcription of defense-related genes in oysters exposed to a harmful alga and its purified toxin.

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Ray's fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR-RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.

Bivalve culture is of considerable economic and social interest in northeastern (NE) Brazil. The polyculture is an alternative approach to traditional monoculture for reducing the environmental impact of shrimp farming and improving oyster culture. Perkinsus marinus andPerkinsus olseni were found infecting oysters in NE Brazil and can threaten oyster production. This study evaluatedPerkinsus spp. occurrence in Crassostrea gasar during all production stages. Oyster spats were produced in a hatchery and grown in shrimp ponds in Rio Grande do Norte state.Perkinsus spp. were surveyed by Ray’s fluid thioglycollate medium and confirmed by polymerase chain reaction. Prevalence and intensity of infection were determined in oysters until they reached 7 cm. Results showed that the broodstock was already infected by Perkinsus (60%), but the derived spats were Perkinsus-free. Oyster spats acquired Perkinsus infection when transferred to ponds. The prevalence gradually increased in the seven months following placement in ponds (73%), and then decreased to 17% by the tenth month. The infections were initially mild, but intensity increased at the final growth stage. In conclusion, it is possible to produce Perkinsus-free C. gasar oyster spats from infected broodstock, and their culture in shrimp ponds is feasible. Resumo O cultivo de bivalves é de grande interesse econômico e social no Nordeste (NE) do Brasil. O policultivo é uma alternativa ao monocultivo tradicional de camarões para reduzir o impacto ambiental e melhorar a produção de ostras.Perkinsus marinus e Perkinsus olseni foram identificados infectando ostras no Nordeste do Brasil e representam uma ameaça a produção de ostras. Este estudo avaliou a ocorrência dePerkinsus spp. em Crassostrea gasardurante todas as fases de produção. Sementes de ostras foram produzidas em laboratório e cultivadas em viveiros de camarão no Rio Grande do Norte.Perkinsus spp. foram diagnosticados com o uso do meio de tioglicolato fluido de Ray e confirmado por reação em cadeia da polimerase. A prevalência e intensidade de infecção foram determinadas em ostras até atingirem 7 cm. Os resultados mostraram que os reprodutores encontravam-se infectados porPerkinsus (60%), mas as sementes produzidas estavam livres de Perkinsus. As sementes adquiriram a infecção porPerkinsus quando transferidas para os viveiros. A prevalência aumentou gradualmente nos sete meses após a colocação nos viveiros (73%) e, em seguida, diminuiu para 17% até o décimo mês. As infecções foram inicialmente leves, mas aumentaram até a fase final do crescimento. Em conclusão, é possível produzir sementes de ostras C. gasarlivres de Perkinsus a partir de reprodutores infectados e seu cultivo em viveiros de camarão é viável.

Aerobic exercise promotes short-term physiological changes in the intestinal smooth muscle associated to the ischemia-reperfusion process; however, few studies have demonstrated its effect on the intestinal contractile function. Thus, this work describes our observations regarding the influence of acute aerobic swimming exercise in the contractile reactivity, oxidative stress, and morphology of rat ileum. Wistar rats were divided into sedentary (SED) and acutely exercised (EX-AC) groups. Animals were acclimated by 10, 10, and 30 min of swimming exercise in intercalated days 1 week before exercise. Then they were submitted to forced swimming for 1 h with a metal of 3% of their body weight attached to their body. Animals were euthanized immediately after the exercise section and the ileum was suspended in organ baths for monitoring isotonic contractions. The analysis of lipid peroxidation was performed in order to determinate the malondialdehyde (MDA) levels as a marker of oxidative stress, and intestinal smooth muscle morphology by histological staining. Cumulative concentration-response curves to KCl were altered in the EX-AC with an increase in both its efficacy and potency (E max = 153.2 ± 2.8%, EC 50 = 1.3 ± 0.1 × 10(-2) M) compared to the SED group (E max = 100%, EC 50 = 1.8 ± 0.1 × 10(-2) M). Interestingly, carbachol had its efficacy and potency reduced in the EX-AC (E max = 67.1 ± 1.4%, EC 50 = 9.8 ± 1.4 × 10(-7) M) compared to the SED group (E max = 100%, EC 50 = 2.0 ± 0.2 × 10(-7) M). The exercise did not alter the MDA levels in the ileum (5.4 ± 0.6 μ mol/mL) in the EX-AC compared to the SED group (8.4 ± 1.7 μ mol/mL). Moreover, neither the circular nor the longitudinal smooth muscle layers thickness were modified by the exercise (66.2 ± 6.0 and 40.2 ± 2.6 μm, respectively), compared to the SED group (61.6 ± 6.4 and 34.8 ± 3.7 μm, respectively). Therefore, the ileum sensitivity to contractile agents is differentially altered by the acute aerobic swimming exercise, without affecting the oxidative stress and the morphology of ileum smooth muscle.

This paper gives an account of the pathogens found in the cultivated Japanese oyster Crassostrea gigas and in the mangrove oyster Crassostrea rhizophorae from natural populations of two sites, Sambaqui (27° 29′18″S, 48° 32′1″W) and Ribeirao da Ilha (27° 42′51″S, 48° 34′6″W), Santa Catarina Island, Brazil. Oysters were collected in March 2008 and April 2009, 150 per site, year and condition (rocky shore and culture). For pathological study, the techniques used were: macroscopic examination, histology and tissue culture in fluid thioglycollate medium specific for Perkinsus . The results showed the presence of the polychaete Polydora sp. with high prevalence (up to 100%) in C. gigas ; gametic hypertrophy in C. rhizophorae from Ribeirao da Ilha and in C . gigas from Sambaqui and Ribeirao da Ilha with low prevalence (3.3%); rickettsia-type bacteria, with greater prevalence in C. gigas (30%) than in C. rhizophorae , causing alteration in the epithelium of the stomach. It was not detected the presence of Perkinsus in any oyster sample analyzed of any site. Ciliates of genus Trichodina were observed among gill lamellae, digestive tubules, and adhered to the gills of oysters from Ribeirao da Ilha, with higher prevalence in C. rhizophorae (50%) and without causing injury. Protozoa Sphenophrya -type was found in the gills of C. gigas and C. rhizophorae , with higher prevalence in C. gigas from Sambaqui (70%), not causing changes in the gills. Protozoa of the genus Ancistrocoma were detected in the digestive tubules of C. gigas (36.7%) and C. rhizophorae (40%) from Sambaqui at low intensity and without causing apparent damage. Steinhausia -type microsporidians were observed in the cytoplasm of oocytes of C. rhizophorae and C. gigas with prevalences up to 33.3%. The intensity of infection in the animals was low, with only one oyster presenting more than 50 oocytes infected. The sporocysts of the pathogen caused alteration in the normal structure of the oocytes cytoplasm. Protozoa of genus Nematopsis were observed in the connective tissue of the gills and mantle with high prevalence (100%) in C. rhizophorae from Sambaqui, without host defense response. The observed metazoan were: turbellarians Urastoma -type, metacestode of genus Tylocephalum and copepods possibly of genus Pseudomiycola , all in low prevalence. None of the pathological occurrences found seems to cause significant damage in oysters, once they were found at low intensities. However, due to the great socioeconomic importance that the shellfish represent to the State of Santa Catarina, it's necessary to keep monitoring the health status of these populations.

Disseminated neoplasia (DN) was one of the most important pathological conditions found in cultured flat oysters (Ostrea edulis) from different geographical origins grow in Galicia (NW Spain), during a two years selective breeding programme to produce oysters less susceptible to bonamiosis. Histological characteristics observed in oysters affected by DN included intense infiltration of connective tissue of various organs (gills, stomach, digestive gland and gonad) by large undifferentiated cells, with a large nucleus and a high nucleus-to-cytoplasm ratio. The main ultrastructural features were predominance of euchromatin over heterochromatin that was arrayed in small clumps in the nucleus, prominent granular nucleolus, swollen mitochondria with few cristae and high number of free ribosomes in the cytoplasm. A seasonal pattern of DN prevalence was detected, with higher values in spring-summer, but there were no significant differences between geographic origins or families within these origins. However, the intensity of the disease was significantly different between origins; oysters originating outside of Galicia (particularly those originating from Ireland) were more susceptible to develop advanced DN. DN (8%) and bonamiosis (4.9%) were found concurrently in oysters. The nature and significance of this association warrants more investigation to determine its importance, if any.

During a parasitological survey of the brown mussel Perna perna from highly productive culture fields in the southern sector of Brazil, monstrilloid copepods were discovered in the mantle of this mytilid bivalve. Numerous specimens of endoparasitic copepods were found within nodules in the mantle of the host; they belong to an undetermined species of Monstrilla. Monstrilloid copepods were known to be endoparasitic in polychaetes and in one species of prosobranch mollusc only; their occurrence as parasites of bivalve molluscs has not been previously documented. This is also the first record of these crustaceans infecting commercial molluscs. The prevalence of Monstrilla sp. infecting the brown mussel was 25.6%. At the histological level, it was observed that the larvae induced a strong haemocytic infiltration resulting from the complete larval encapsulation within the host mantle. The effect of this monstrilloid in the cultured populations of P. perna deserves further study.

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum . Manila clams, with variable levels of infection with Perkinsus olseni , were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum . After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum ; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.

An experiment was carried out to compare the performance of various cohorts of oysters Ostrea edulis from each of four different geographic origins that were born and cultured in the same environment. This paper reports the variation of gonad condition of the oysters through on growing and analyses of the relationship between gametogenesis and infection by Bonamia ostreae. Nineteen cohorts of spat were produced in a hatchery from four oyster populations: Irish, Greek, and two Galician: Ortigueira and Coroso; the spat were transferred to a raft in the Ria de Arousa, an area affected by bonamiosis, for grow-out for two years. Significant differences were observed in the temporal pattern of gonad condition between oysters from different geographic origins. Irish oysters showed a short period of gonad activity and a long gonad resting phase. By contrast, Greek oysters had the longest period of gonad activity and the shortest gonad resting phase, as well as the highest percentage of male oysters compared to oysters from other origins. Galician oysters, from both origins, showed a pattern of gonad development intermediate between Irish and Greek, with a higher percentage of females during the second reproductive period. Significant differences were also observed between cohorts within Greek, Coroso and Irish origin, but not from Ortigueira, whose cohorts showed a similar temporal pattern of gonad condition. The differences between oysters from different origins would indicate adaptation to different environments; nevertheless the significant differences that were found between cohorts within origins would indicate that there is variability that eventually could allow adaptation to new environments. Considering sex categories, heavy infections by B. ostreae were significantly more frequent in oysters with a predominant female component; considering gonad stage, heavy infections were significantly more frequent in ripe and partially spawned oysters. The results suggest a hypothetical enhancement of progression of infection by B. ostreae associated with female maturation.

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ria de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.

In the present study, we evaluated the influence of chronic swimming aerobic exercise on the contractile reactivity, lipid peroxidation and tissue morphology of small intestine from rats. We showed that, accordingly to the time of exercise, the rat ileum has its contractile reactivity reduced, in addition to the increased lipid peroxidation followed by its reestablishment to basal levels, as well as the augmented longitudinal smooth muscle layer thickness together with the reduced circular thickness., Several studies have reported the gastrointestinal (GI) effects promoted by the physical exercise. Thus, we aimed to evaluate the influence of swimming exercise on the contractile reactivity, lipid peroxidation and morphology of rat ileum. Wistar rats were divided into sedentary (SED) and groups exercised for two (EX2), four (EX4), six (EX6) or eight (EX8) weeks, 5 days/week. Animals were killed; the ileum was removed and suspended in organ baths where the isotonic contractions were recorded. Lipid peroxidation was evaluated by MDA (malondialdehyde) measurement with TBARS (thiobarbituric acid reactive substances) assay and morphology by histological staining. Cumulative concentration-response curves to KCl were attenuated, as the Emax values were changed from 100% (SED) to 63.1±3.9 (EX2), 48.8±3.8 (EX4), 19.4±1.8 (EX6) and 59.4±2.8% (EX8). Similarly, cumulative concentration-response curves to carbamylcholine hydrochloride (CCh) were attenuated, as the Emax values were changed from 100% (SED) to 74.1±5.4 (EX2), 75.9±5.2 (EX4) and 62.9±4.6 (EX6), but not in the EX8 (89.7±3.4%). However, CCh potency was increased in this latter, as the EC50 was altered from 1.0±0.1×10−6 (SED) to 2.1±0.4×10−7 (EX8). MDA concentration was altered only in EX4 (44.3±4.4) compared with SED (20.6±3.6 μmol/l). Circular layer was reduced in SED when compared with the exercised groups. Conversely, longitudinal layer was increased. In conclusion, chronic swimming exercise reduces the ileum contraction, equilibrates the oxidative damage and promotes changes in tissue size to establish an adaptation to the exercise.

SUMMARYThe oyster Crassostrea gasar is a species widely used as food and a source of income for the local population of the estuaries of Northeast Brazil. Perkinsus marinus and Perkinsus olseni are deleterious parasites for oyster farming and were recently detected in Brazil. In this study, a histopathologic survey of the oyster C. gasar cultured in the estuary of the River Mamanguape (Paraíba State) was performed. Adult oysters were collected in December 2011 and March, May, August and October 2012 and processed for histology and Perkinsus sp. identification by molecular analyses. Histopathological analysis revealed the presence of parasitic organisms including viral gametocytic hypertrophy, prokaryote-like colonies, protozoans (Perkinsus sp. and Nematopsis sp.) and metazoans (Tylocephalum sp. and cestodes). Other commensal organisms were also detected (the protozoan Ancistrocoma sp. and the turbellarian Urastoma sp.). The protozoan parasite Perkinsus sp. had the highest overall prevalence among the symbiotic organisms studied (48·9%), followed by Nematopsis sp. (36·3%). The other organisms were only sporadically observed. Only the protozoan Perkinsus sp. caused alterations in the oysters’ infected organs. Molecular analyses confirmed the presence of P. marinus, P. olseni and Perkinsus beihaiensis infecting the oyster C. gasar. This is the first report of P. beihaiensis in this oyster species.

International audience; Species of the dinoflagellate genus Alexandrium produce phycotoxins responsible for paralytic shellfish poisoning. Blooms of Alexandrium minutum reach very high concentrations of vegetative cells in the water column; and when these blooms occur, large numbers of toxic cysts can be produced and deposited on sediments becoming available to benthic species. The present study investigated the potential effect of exposure to toxic cysts of A. minutum on the periwinkle Littorina littorea. Snails were exposed for nine days to pellicle cysts of toxic and non-toxic dinoflagellates, A. minutum and Heterocapsa triquetra, respectively, followed by six days of depuration while they were fed only H. triquetra. Toxin accumulation, condition index, immune and histopathological responses were analyzed. Histological alterations were also monitored in snails exposed to a harmful A. minutum bloom, which naturally occurred in the Bay of Brest. Snails exposed to toxic cysts showed abnormal behavior that seems to be toxin-induced and possibly related to muscle paralysis. Periwinkles accumulated toxins by preying on toxic cysts and accumulation appeared dependent on the time of exposure, increasing during intoxication period but tending to stabilize during depuration period. Toxic exposure also seemed to negatively affect hemocyte viability and functions, as ROS production and phagocytosis. Histological analyses revealed that toxic exposure induced damages on digestive organs of snails, both in laboratory and natural systems. This study demonstrates that an exposure to the toxic dinoflagellate A. minutum leads to sublethal effects on L. littorea, which may alter individual fitness and increase the susceptibility of snails to pathogens and diseases.

This study reports the prevalence of Bucephalus sp. in Perna perna populations from a culture station of southern Brazil and its effect on the mussel reproductive tissue and immune system. The prevalence of Bucephalus sp. in P. perna (n = 1871) was considered low (3.1%) and did not seasonally vary. Histological sections of the mantle of infected mussels revealed a marked (80%) reduction of the reproductive tissue that was severe even in mussels exhibiting a moderate infection degree. The total (THC) and differential (DHC) hemocyte counts were lower in infected mussels (3.9 x 10(6) hem/ml; granular hemocytes = 33%) as compared with non-infected animals (5.5 x 10(6) hem/ml; granular hemocytes = 40%). The plasma protein concentration did not vary upon infection. Hemocyte infiltration was significantly higher only in mussels with a very heavy infection degree. The parasite sporocysts were never seen encapsulated by the host hemocytes. Our results indicate that Bucephalus sp. promotes a severe castration in its host and apparently evades the mussel immune system.

International audience; Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection ("Light-Moderate" and "Moderate-Heavy"). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index.

This is the first report of Perkinsus sp. (Bivalvia: Veneridae) infecting wild clams of the species Anomalocardia brasiliana in Brazil. The gill lamellae and rectum of 150 specimens of A. brasiliana collected in the Timonha river estuary (Ceara, Northeastern Brazil) in March 2012 were incubated in Ray's fluid thioglycollate medium (RFTM) for detection of Perkinsus sp. In RFTM, the prevalence of Perkinsus sp. was 14.7% (22/150) and the intensity of infection ranged from very light (1-10 cells across the slide) to light (12-100 cells). The presence of Perkinsus sp. was confirmed by PCR in seven (31.8%) out of 22 RFTM-positive specimens. DNA sequencing confirmed the presence of the genus Perkinsus and the phylogenetic analysis strongly indicated Perkinsus beihaiensis as the species responsible for the infection.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceara state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraiba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio Sao Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

This study investigated the health of natural stocks of the oysterCrassostrea rhizophorae on the southern coast of Bahia in northeastern Brazil, during summer and winter 2010, at three localities (sampling points) in the estuaries of the Maraú (Camamu Bay) and Graciosa rivers. A total of 180 oysters (30/sampling point/season) were examined macroscopically for the presence of pathogens and anatomical changes. The specimens were subsequently fixed in Davidson solution, processed for paraffin embedding, sectioned and stained with Harris' hematoxylin and eosin. Histological analysis revealed the presence of Rickettsia-like organisms (RLOs), Ancistrocoma,Trichodina, Sphenophrya,Nematopsis, Urastoma,Bucephalus in the sporocyst phase, a nonspecific metacercaria, and a metacestode of genus Tylocephalum. The prevalence of infection was low except for parasitism byNematopsis sp. which also caused histopathological changes. The presence of Bucephalus sp. caused parasitic castration. These two pathogens significantly affect the health of C. rhizophorae. Este estudo investigou a saúde de ostras da espécieCrassostrea rhizophorae de estoques naturais do Litoral Sul do Estado da Bahia, Nordeste do Brasil, durante o verão e o inverno de 2010, em três pontos amostrais distribuídos nos estuários dos rios Maraú (Baía de Camamu) e Graciosa. Um total de 180 ostras (30/ponto amostral/período) foram examinadas macroscopicamente para a presença de patógenos e alterações anatômicas e posteriormente fixadas em solução de Davidson, processadas para inclusão em parafina, seccionadas e coradas com hematoxilina de Harris e eosina. A análise histológica evidenciou a presença de organismos com características similares a Rickettsia(RLOs), Ancistrocoma, Trichodina,Sphenophrya, Nematopsis,Urastoma, Bucephalus em fase esporocística, metacercária inespecífica e metacestóide de Tylocephalum. As prevalências de infecção foram baixas, com exceção do parasitismo por Nematopsis sp., o qual também causou alterações histopatológicas. A presença deBucephalus sp. causou castração parasitária. Esses dois patógenos têm interferência significativa na saúde de C. rhizophorae.

International audience; Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases.

This study investigated the occurrence of the protozoan Perkinsus in the oyster Crassostrea rhizophorae on the coast of Bahia State, Brazil. The oysters (n=900) were collected in February–March and July–August 2010. The Ray’s fluid thioglycollate medium (RFTM) analysis of gills and rectum revealed hypnospores of Perkinsus sp. with a high mean prevalence (63%). The infection intensity varied from very light to advanced. The polymerase chain reaction confirmed Perkinsus in 87.2% of the RFTM-positive oysters. Histological analysis showed trophozoites and schizonts phagocytized by hemocytes, mainly in the intestine and the stomach epithelium.